Keratoconus is a visually disabling disease characterized by a biomechanical failure of the cornea and it affects patients since their adolescence. The specific mechanisms causing the disease are not fully understood so no pharmacological treatment is currently available. Additionally, there are no available biomarkers for rapidly progressive disease that occurs in 25% of the patients. Loss of collagen type XII and I is a hallmark of keratoconus. Moreover, our preliminary data and the existing literature suggest that the Wnt signaling pathway is dysregulated in keratoconus epithelium. This is of interest because Wnt signaling is known to regulate extracellular matrix production. Additionally, our preliminary data shows that Wnt signaling positively regulates collagen type XII transcription. This particular collagen, that is known to interact with type I collagen, is thought to impart mechanical strength to stress bearing structures. We hypothesize that collagen type XII depletion resulting from Wnt signaling dysregulation contributes to the biomechanical instability in keratoconus. Additionally, WNT10A and COL12A1 expression levels in the epithelium of progressive keratoconus patients are correlated with specific corneal tomographical features, so they may serve as biomarkers of progressive keratoconus. Three specific aims are proposed to elucidate disease mechanisms in the corneal epithelium of keratoconus patients: 1) COL12A1 perturbed immortalized corneal epithelial cells (hTCEpi) will be grown on 3D collagen matrices and tensile strength will be assessed. The gels will also be examined for morphological changes using electron microscopy. 2) Wnt signaling will be perturbed in hTCEpi cells grown in culture and the effect on COL12A1 and COL1A1/2 will be assessed through gain and loss of function models using stable transfection techniques. Additionally, WNT10A null mouse corneas will be examined for changes in morphology, COL12A1 and COL1A1/2 transcript expression and protein production. 3) WNT10A and COL12A1 expression in keratoconus corneal epithelium will be assessed in a large cohort of keratoconus patients undergoing corneal collagen cross?linking. The expression levels will be correlated with clinical markers of progression. The ultimate goal of this application is to uncover specific disease mechanisms in the keratoconus corneal epithelium in order to develop new treatments for this disease. Additionally, we aim to identify biomarkers of progressive disease.